In the experimental traumatic brain injury model (TBI), the number of UT-B positive neurons near the ipsilateral cerebral cortex increased first and then decreased over time, peaking at the 24 h. We inferred that change in UT-B expression after the TBI was an adaptation to changed urea levels. The experimental data suggest that the UT-B may be a potential target for the treatment of TBI and white matter edema.Urea transporter B (UT-B) is a membrane channel protein widely distributed in mammals, and plays a significant physiological role by regulating urea and water transportation in different tissues. More and more studies have found that UT-B is related to neurological diseases, including myelinopathy and depression. When urea accumulates in the brains of UT-B knockout mice, the synaptic plasticity of neurons is reduced, and the morphology and function of glial cells are also changed. However, the distribution and expression change of UT-B remain unclear. The purpose of this study is to determine the expression characteristics of UT-B in the brain. Through single-cell RNA sequencing, UT-B was found to express universally and substantially throughout the various cells in the central nervous system except for endothelial and smooth muscle cells. UT-B was detected in the third cerebral ventricular wall, granule cell layer of the dentate gyrus, and other parts of the hippocampal, cerebral cortex, substantia nigra, habenular, and lateral hypothalamic nucleus by immunohistochemistry. Compared with the membrane expression of UT-B in glial cells, the subcellular localization of UT-B is in the Golgi apparatus of neurons. Further, the expression of UT-B was regulated by osmotic pressure  Slc14a1 gene, has been documented in various tissues including kidney, ureter, bladder, testis, erythrocyte, fetal liver, spleen, bone marrow, intestine, colon, heart, aorta, cochlea, and brain  are a group of membrane channel proteins facilitating the massive and rapid movement of urea through different tissues Sands, . Urea trThe UT-B is widely distributed in the central nervous system. UT-B protein was found in astrocyte endfeet processes surrounding microvessels and ependymal cells lining along cerebral ventricles  to determine the expression of UT-B in 4,884 cells from the wild-type mouse brain and analyzed the expression ratio of UT-B in each cell subtype. The distribution of UT-B in rat brain regions was detected by immunohistochemistry. Whether in the mouse, rat, or human brain, we confirmed the expression of UT-B in neurons. We also found the subcellular location and expression level of UT-B were regulated by osmotic pressure in cultured cortical neurons. UT-B expression level changed in the rat brain trauma over time. These data uncovered a detailed description of the UT-B expression in the central nervous system. Furthermore, the expression characteristics of UT-B regulated by osmotic pressure suggest that it may be involved in the pathological progression of brain edema and further research is needed to elucidate thoroughly.Adult male Sprague-Dawley rats in the weight range of 220–250 g obtained from the Animal Breeding Facility of Chongqing Medical University were prepared for the experiments. Rats (n = 30) were divided into five groups based on time intervals after traumatic brain injury . Animal studies were conducted following the procedure approved by the Animal Ethics Committee of Peking University. The approval number is LA2020354.ad libitum. All protocols involving mice were approved by the Institutional Animal Care and Use Committee at the Peking University Health Science Center .C57BL/6J male and female mice (7- to 8-week-old) were acclimated to Peking University, Beijing, vivarium for at least 7 days before experiments. Food and water were available UT-B knockout mice were generated by crossing C57BL/6J background germline-heterozygous-null mutant UT-B knockout mice as described  prepared previously. After taking the brain tissue out of the skull, a mouse brain slice mold was used to cut into 1-mm brain slices. The brain tissues were then digested using ACSF diluted papain at 37°C for 30 min and were mixed every 10 min After filtering with a 400-mesh cell sieve, the suspension was diluted with 50 ml ACSF at 4°C. After 30-min enzymatic digestion at 37°C with shaking of the tube every 10 min, the suspensions were filtered through an ACSF-equilibrated 35-mm cell strainer. After filtering, the suspension was diluted in a large-volume  ice-cold ACSF, followed by centrifugation  to reduce debris. The supernatant is then carefully discarded. The pelleted cells were resuspended in 200 μl of dulbecco's modified eagle medium (DMEM) containing 1% bovine serum albumin (BSA). The 10 × genomics chromium single-cell kit was then used to perform scRNA-seq detection on the suspension.Artificial cerebrospinal fluid was prepared as follows: 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaHThe experimental traumatic brain injury model was created in the right hemisphere referred to Feeney's method  and chilled on ice. The dissected medial prefrontal cortex was centrifuged  after dispersed by trituration and 0.25% trypsin digestion . The culture medium was DMEM  supplemented to 10% FBS  for the first 4 h, then changed to neurobasal  with 2% B27  and 0.5 mM L-glutamine . Cells were plated on 0.1% poly-L-lysine  coated 6-well plates at 1 × 106 cells/ml of medium, and maintained in an incubator at 37°C and 5% CO2. Urea , mannitol , and sucrose  were dissolved into the medium for stimulation. Neurons were subjected to subsequent experiments after 9 days culture.Newly postnatal C57BL/6J mice were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center, Beijing. Anesthetized mice were killed by cervical dislocation. All tissues were maintained in D-Hank's solution  and perfusion with 20 ml of 0.9% saline, followed by fresh 4% paraformaldehyde in 0.01 M phosphate-buffered saline (pH 7.3). After perfusion, brains were taken out for an additional fixation overnight in 4% paraformaldehyde, followed by a graded series of sucrose-gradient dehydration. Dehydrated samples were frozen and sectioned in the coronal plane at 20 μm for immunofluorescent detection . The human brain slices were paraffin sections and needed antigen retrieval. Slides were processed for antigen retrieval and were dipped in 0.1 M sodium citrate buffer (pH 6.0) and heated to boiling for 5 min twice in a Microwave oven and left to return to ambient temperature. The brain slices were subsequently washed three times by phosphate buffer saline (PBS) for 5 min. Then the slices were washed in PBS–T (0.3% Triton X−100 in PBS) for 0.5 h at 37°C and transferred to blocking solution (0.3% PBS–T in 5% goat serum) overnight at 4°C before ~36 h incubation (0.3% PBS–T in 1% serum) in primary antibody solution [rabbit anti–UT-B with 1:100 working dilution, a gift from Dr. Trinh-Trang-Tan MM  and heated to boiling for 5 min twice in a microwave oven and left to return to ambient temperature. And then the sections were incubated in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. Followed by incubation in 10% goat serum, the sections were incubated overnight at 4°C with the primary rabbit polyclonal anti-UT-B antibody with 1:500 working dilution. After rinsing with phosphate buffer solution for 5 min thrice, the sections were performed with a sensitive polymer-helper detection system  following the manufacturer's instruction. Diaminobenzidine was used for staining development and the sections were counterstained with hematoxylin and then examined by light microscopy. Negative controls consisted of substituting normal serum for primary antibodies. The mean optical density of immunostained slides for UT-B was analyzed using the Image-Pro Plus software . The slides were scanned using 20 × magnification. By comparing each time point, the tendency variations of UT-B expression can be obtained.Slc14a1, we identified that Slc14a1 was expressed in most cells except endothelial and smooth muscle cells  but not ineffective osmoles (urea) in isolated perfused tubules (Sands and Schrader, AQP4 deletion alleviates cytotoxic brain edema but aggravates vasogenic brain edema (Papadopoulos and Verkman, TBI can cause activation of excitatory amino acids, calcium overload, mitochondrial damage, activation of oxygen free radicals, activation of inflammatory system, etc. (Desai and Jain, In conclusion, we found that UT-B is selectively expressed in the central nervous system glial cells and neurons; and the translocation of UT-B in hypo- or hyperosmolar conditions. The expression is highly consistent in mice, rats, and humans, proving that UT-B is a very conservative urea transporter. We revealed that UT-B changed among time after TBI, and the turning point is 24 h after injury, which may be adaptive adjustments to the changed urea levels. However, the molecular mechanism by which UT-B exerts specific effects in various brain regions and post-traumatic brain injury is unclear. Hence, further research is needed to thoroughly elucidate the role and mechanism of UT-B in the central nervous system.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Second Affiliated Hospital of Chongqing Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by Animal studies were conducted in accordance with the procedure approved by the Animal Ethics Committee of Peking University, the approval number is LA2020354.BH, HW, JR, and BY conceptualized the project. BH and HW were involved in designing and conducting the experiments. DZ and JM assisted with cell culture. ML performed part of animal experiments. BH analyzed data and prepared the draft with input from all authors. JR and BY supervised research. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, a crosslinked polycarboxylate superplasticizer (crosslinked-PC) was synthesized via the free radical polymerization reaction. Pentaerythritol tetraacrylate (PETA) was used as the crosslinked agent. A comparative comb-like polycarboxylate superplasticizer (comb-like-PC) was prepared under the same reaction conditions. The dispersion retention capacity, dispersion capability, hydration characteristics of the cement paste and setting time were investigated in detail. At the dosage of 0.6% bwoc, the fluidity of the cement/crosslinked-PC paste was about 340 mm, which was 40~50 mm larger than the cement/comb-like-PC paste. The dispersion retention capacity of the cement/crosslinked-PC paste was observed to be much superior due to higher adsorbed amounts on the cement particles. Moreover, the cement/crosslinked-PC paste exhibited the initial and final setting durations of 196 and 356 min, respectively, which indicated an enhancement of 18 and 68 min compared to the cement/comb-like paste. The crosslinked copolymers exhibit a stronger retardation effect than the comb-like copolymers due to their enhanced adsorbed amounts and stronger steric hindrance effect. This is further illustrated by the characterization of the hydration process and hydration products. It can be concluded that it is feasible to improve the dispersive capacity and the dispersion retention capacity of PC by changing the molecule structure from comb-like to slightly crosslinked. The polycarboxylic superplasticizers (PCs) have been widely applied in the field of commercial concrete. As China has emerged as the world’s largest construction market, it has also boosted the application of superplasticizers. PCs have become the essential components of concrete owing to its advantages such as its low dosage, notable dispersive and dispersion retention capacity, environmental friendliness and designability of molecular structure ,3,4,5,6.With the advancements in molecular structure diversification, the molecular structure design for PCs has become a focus of research for attaining a superior performance. A number of research studies have reported the synthesis of novel PCs with the crosslinked molecular structures, with much improved performance. Etsuo et al. controlled slump loss by introducing the diethylene glycol diacrylate as a crosslinking agent in PC copolymers . Cai et In this work, a crosslinked polycarboxylate superplasticizer (crosslinked-PC) was synthesized. Pentaerythritol tetraacrylate (PETA) was used as the crosslinked agent. The dispersion maintaining ability, dispersion capability, hydration characteristics of the cement paste and setting time were investigated in detail. Additionally, the adsorption behavior of crosslinked-PC was investigated by measuring the adsorption amount, and the working mechanism of the crosslinked-PC in the cementitious system was further summarized.w = 2200 g/mol) was provided by Taijie Chemical Co., Ltd. . The cement (P.I 42.5) was supplied by China United Cement Corporation . AA, HPEG, SAMS and TGA were added to a flask and dissolved in 40 mL deionized water through the continuous agitation reactor. The crosslinker PETA and initiator KPS were mixed completely in deionized water (30 mL) and then added to the flask by peristaltic pump at a rate of 0.5 mL/min. The polymerization was carried out at 75 °C for four hours. The prepared sample was denoted as crosslinked-PC. A comparative sample was performed in the absence of PETA under the same reaction conditions and which was denoted as comb-like-PC. The aqueous products were neutralized to pH = 7 by NaOH solution. The synthetic routes of the crosslinked-PC and comb-like-PC are shown in Materials Pentaerythritol tetraacrylate (PETA), acrylic acid (AA), potassium peroxodisulfate (KPS), sodium methylallyl sulphonate (SAMS) and thioglycolic acid (TGA) were provided by Aladdin Industrial Corporation . The methyl allyl polyethylene glycol (HPEG) (MMethods Fourier Transform Infrared (FTIR) Measurement. FTIR spectra were measured with an FTIR spectrometer . The spectra were recorded with a spectral range of 400~4000 cm−1. All samples were dried at 60 °C for 12 h in a vacuum oven before FTIR measurements.Gel Permeation Chromatography (GPC) Measurement. The molecular weights were measured with Waters 1525/2414 instrument . PEG was used as the calibration standard, the measurement was performed at 25 °C.1H-NMR Measurement. The 1H-NMR results were measured with a 400 MHz DRX-400 spectrometer . Samples for measurement were dissolved in deuteroxide. The chemical shift values were expressed in δ values (ppm) relative to tetramethylsilane (TMS) as an internal standard.Cement Paste Fluidity Measurement. Cement paste fluidity test was measured depending on the standardization of GB/T 8077-2012 [077-2012 .Total organic carbon (TOC) Measurement. The adsorbed amounts of the PCs on cement particles were measured with a TOC analyzer . In total, 10 g blank cement was mixed with superplasticizer solution , respectively. Then, the cement paste was centrifuged at 5000 rpm for 10 min. The supernatant solution was diluted with deionized water for TOC measurement. Each sample was tested thrice, the average value was calculated.Setting Time. The initial and final setting time of cement pastes were determined by a Vicat analyzer depending on the standard GB/T 1346-2011 [346-2011 . The iniIsothermal Calorimetry Measurement. The hydration heat was measured with an isothermal conduction calorimeter for 7 days. In total, 10.0 g cement was filled into 20 mL glass ampoules, mixed with the PC solution (w/c = 0.4).Rheological Measurements. Rheometric measurement of cement pastes were performed by a DHR Rheometer  using a vane fixture to assess changes in paste viscosity.X-ray Diffraction (XRD) Measurement. X-ray diffraction was measured with a D8 ADVANCE power diffractometer , the diffraction angle (2θ) was from 2 to 50°.Compressive Strength Measurement. Compressive strength measurements were determined by a TYE-2000B machine. The cement mixture (w/c = 0.35) was fed into a 7 cm × 7 cm × 7 cm mold. The cement block was hydrated for 7 days.Scanning electron microscope (SEM) Measurement. The morphology of the hydration products was measured with a scanning electron microscope . Ethanol was utilized to discontinue the hydration process.−1 attributed to the C-O-C vibration and stretching vibration of polyethylene glycol, respectively [−1 corresponded to the binding of -C=O for carboxylic acid. The peak at 3450 cm−1 was attributed to the stretching vibration of the -OH and the signals at 1470 cm−1 were due to C-H bending vibrations, respectively [ectively . Furtherectively . These r1H-NMR spectra of the comb-like-PC and crosslinked-PC. For all samples, the peaks between 3.4~3.8 and 4.5~5.0 ppm corresponded to the H atoms of polyethylene glycol [2- in crosslinker PETA but no peak at this position for comb-like-PC. Furthermore, the characteristic peak (b) at 1.84 ppm in crosslinked-PC also does not exist in comb-like-PC, which belonged to the H atoms of double bonds in PETAs’ structure. The aforementioned characteristic peaks of the H atoms in PETA demonstrated that the crosslinker was introduced to the crosslinked-PC successfully.e glycol . Crossli1H-NMR and GPC results demonstrated that the PCs with different structures were prepared successfully.The molecular weight of comb-like-PC and crosslinked-PC were analyzed by GPC measurement. The respective GPC curves are displayed in 2, cement suspension shows strong alkalinity. Thus, it can be inferred that at the initial stage of hydration, the ester groups from crosslinker PETA could be hydrolyzed into carboxylate radical in the cement suspension. As shown in To evaluate the performance of the superplasticizers, the dispersion retention capacity is another important factor. Dispersion retention capacity is defined as the decrease in fluidity over time. Thus, the change in fluidity was recorded over 2 h. As shown in The setting time is directly related to the cement hydration process . As soonThe exothermic heat flow curves of the hydration process are shown in tA, the end point of the acceleration period was denoted as tC, and the point of the maximum acceleration rate was denoted as tB. Meanwhile, the heat generation rate during the induction period was denoted as (dQ/dt)A, and the maximum hydration rate in the acceleration period was denoted as (dQ/dt)C, respectively. kA–B corresponds to the secant slope on the exothermic heat flow curve between A and B, which means the acceleration rate in the acceleration period. As noted in A–B and (dQ/dt)C decreased on the addition of the comb-like-PC and crosslinked-PC. Moreover, the comb-like-PC led to a weaker retardation effect than the crosslinked-PC, thus, suggesting that crosslinked-PC had a strong impact on delaying the generation of the hydrates during the acceleration period. Thus, the crosslinked copolymers exhibit a stronger retardation effect than the comb-like copolymers due to their enhanced adsorption amounts and strong steric hindrance effect.The parameters of the hydration kinetics were obtained from the exothermic heat flow curves in η’ of cement pastes (w/c = 0.35) with the PC dosage of 0.5% bwoc. A shear thinning behavior was observed for all samples from a shear rate of 0.01~50 rad/s. With a dosage of 0.5% bwoc, the cement paste containing cement/comb-like-PC and cement/crosslinked-PC had a viscosity at 0.01 rad/s of 37.6 and 23.5 Pa s, respectively. Cement paste had the highest viscosity at 0.01 rad/s at 105.2 Pa s, which is about 3 and 4 times than that of cement/comb-like-PC and cement/crosslinked-PC paste, respectively. This suggests that the crosslinked-PC led to a more significant sharp reduction in yield stress than comb-like-PC, which is consistent with the results of fluidity. With the increase in shear rate, a clear shear thinning behavior for the pastes occurred. Thus, the addition of crosslinked-PC sharply decreased the aggregation of cement pastes and resulted in the transition from the solid-like to liquid-like viscoelastic behaviors.The addition of PC has an influence on the rheological response of cement paste . Figure 4AF), dicalcium silicate (C2S), tricalcium aluminate (C3A) and tricalcium silicate (C3S). The hydration rates of C3S, C3A and C4AF are faster than C2S [2 crystals and amorphous C-S-H gel. The XRD patterns of cement hydration products are shown in o occurred due to the formation of the Ca(OH)2 crystals [o, while the signal in cement/crosslinked-PC could hardly be observed, which indicated that the addition of PCs delayed the hydration process. For the XRD patterns of the hydration products after being hydrated for 7 days, the differences in XRD patterns could hardly be distinguished; thus implying that the retardation effect of hydration in the case of the crosslinked-PC was mainly reflected during the early stages. Compared to the hydration products hydrated for 1 day, due to the conversion from the AFt phase to the AF-mono (AFm) phase, the diffraction peaks corresponding to the AFt phase were decreased, and the diffraction signals of Ca(OH)2 became strong after hydration [The main components of cement are tetracalcium aluminoferrite 2 crystals. During the early hydration stage, the hydration products were needle-like AFt phase, square Ca(OH)2 crystal and amorphous C-S-H gel. The cement slurry was in a plastic state, and the porosity was largely retained. However, the crystals were too small to connect with each other to result in a steady state. Comparing the effect of the comb-like-PC and crosslinked-PC on the cement hydration process, it could be concluded that the crosslinked-PC retarded the hydration of cement and provided a dense structure during the hydration process. After hydration for 7 days (r 7 days a’–c’, thIn this study, a pentaerythritol tetraacrylate-based crosslinked-PC has been successful synthesized. In comparison with the comb-like-PC, the crosslinked-PC exhibited a higher dispersive capacity. At the dosage of 0.6% bwoc, the fluidity of the cement/crosslinked-PC paste was about 340 mm, which was 40~50 mm larger than the cement/comb-like-PC paste. The dispersion retention capacity of the cement/crosslinked-PC paste was observed to be much superior due to higher adsorbed amounts on the cement particles. Moreover, the cement/crosslinked-PC paste exhibited the initial and final setting durations of 196 and 356 min, respectively, which indicated an enhancement of 18 and 68 min compared to the cement/comb-like paste. The crosslinked copolymers exhibit a stronger retardation effect than the comb-like copolymers due to their enhanced adsorbed amounts and stronger steric hindrance effect. Moreover, the compressive strengths of the cement/crosslinked-PC were similar and the highest compressive strength of 60 MPa was obtained at the dosage of 0.2% bwoc. Thus, it is feasible to improve the dispersive capacity and the dispersion retention capacity of PC by changing the molecular structure from comb-like to slightly crosslinked. The findings reported in this study exhibit the potential for expanding the preparation method of the PCs.


Immune checkpoint inhibitors (ICIs) are increasingly used in the treatment of non-small cell lung cancer (NSCLC). Most randomized clinical trials have excluded patients with brain metastasis (BM), and real-life patients with NSCLC who receive ICIs are not routinely scanned with magnetic resonance imaging (MR-C) of the brain prior to ICI. This means that there are no prospective data available on the prevalence of BM or on the rate of intracranial response (ICR) attributable to ICIs. To evaluate this along with the impact of BM on quality of life and overall survival, we used MR-C as a screening tool in 159 ICI-eligible patients with advanced NSCLC prior to first ICI. At the time of ICI initiation, 28% of patients had BM. Of those who received ICI without additional early local radiotherapy or surgery, 50% had intracranial response at their first MR-C assessment. Long-term survival of patients with BM was comparable to those without.Sparse data exist on immune checkpoint inhibition (ICI) in NSCLC patients with brain metastasis (BM), especially for those with no local therapy (LT) (whole brain radiation therapy (WBRT), stereotactic RT (SRT) or neurosurgery) preceding ICI. Our aims were to investigate the prevalence of BM, rate of intracranial response (ICR), and survival and quality of life (QoL) in real-life patients with advanced NSCLC undergoing palliative ICI. This was a prospective non-randomized study (NCT03870464) with magnetic resonance imaging of the brain (MR-C) performed at baseline resulting in a clinical decision to administer LT or not. ICR evaluation (MR-C) at week 8–9 (mRECIST criteria) for group A (LT) and group B (untreated) was assessed. Change in QoL was assessed using EQ-5D-5L. Of 159 included patients, 45 (28%) had baseline BM. Median follow-up was 23.2 months (IQR 16.4–30.2). Of patients in group A (21) and B (16), 16/37 (43%) had symptomatic BM. ICR was 8/21, 38%  for group A versus 8/16, 50% for group B. No statistical difference in median overall survival of patients with BM (group A: 12.3 (5.2-NR), group B: 20.5 months (4.9-NR)) and without (22.4 months (95% 16.2–26.3)) was obtained. Baseline QoL was comparable regardless of BM, but an improved QoL (at week 9) was found in those without BM. Patients with NSCLC and BM receiving ICI had long-term survival comparable to those without BM. Most patients with newly diagnosed non-small cell lung cancer (NSCLC) present with metastatic disease and are thus eligible for palliative treatment only. Approximately 40% of patients with NSCLC will develop brain metastasis (BM) ,2,3, whiFor patients with NSCLC, different approaches for obtaining intracranial disease control have evolved within the last decade with regard to both local and systemic therapies. Local therapies include stereotactic radiotherapy (SRT), whole brain radiotherapy (WBRT) and neurosurgery ). New and important systemic therapies include the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib, and the next- and third-generation anaplastic lymphoma kinase (ALK) inhibitors alectinib, brigatinib and lorlatinib ,9,10,11.Immune checkpoint inhibition (ICI) has changed the clinical treatment paradigm for patients with NSCLC especially within the last 5 years. ICI is increasingly used alone or in combination with other modalities such as chemotherapy and/or radiotherapy. Data for the subgroup of patients with BM from NSCLC are sparse, since most randomized clinical trials (RCTs) of ICI have excluded patients with BM, or only included patients with stable, previously treated, and asymptomatic BM not requiring corticosteroids (CS) ,16,17,18Since baseline MR-C is not routinely performed outside clinical trials for patients with NSCLC receiving ICI with palliative intent , the freThe primary endpoint of this prospective study was to determine the prevalence of BM at the time of first ICI in a consecutive group of patients with advanced NSCLC regardless of baseline CNS symptoms. Secondary endpoints were rate and duration of intracranial response for patients with BM, overall survival (OS) and QoL.ClinicalTrials.gov identifier (NC T03870464).This was a prospective cohort study including patients with advanced NSCLC eligible for ICI. Key eligibility criteria were histologically verified NSCLC, advanced stage III-IV , age ≥ 18 years, clinically suitable for ICI according to Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0–2, and able to follow study procedures. Patients with autoimmune disease were eligible. A maximum of 10 mg of prednisolone or equivalent corticosteroid (CS) based medication was allowed at inclusion and during the ICI course. No exclusion was made based on prior therapy. Patients were included within 1 week prior to first ICI, and the index date was the date of first ICI.Based on the results of MR-C screening , patients were divided into two major groups—those without BM and those with BM. The group with BM was further divided into two groups based on the addition of local therapy for BM or not. Group A: Patients with BM, who had received local therapy within the last 4 weeks prior to first ICI or during the first 6 weeks post first ICI. Group B: Patients with untreated BM, who did not receive local therapy during the first 6 weeks post first ICI. Patients with BM, who had received local therapy more than 4 weeks prior to first ICI, were included in the group with BM but neither part of group A or B . A certified pathologist determined programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) in extra-cranial tumor tissue by immunohistochemistry using PD-L1 IHC 22C3 pharmDx kit. EGFR and ALK fusion oncogene testing were performed in patients with non-squamous cell carcinoma at the time of primary lung cancer diagnosis, or in cases with recurrent disease through a biopsy just preceding first ICI. Administration of ICI in both first and subsequent treatment lines was based on recommendations from the Danish Medicines Council at the particular time-point during the recruitment period . For dosThe primary endpoint was to determine the proportion of patients with BM at time of first ICI in a consecutive group of real-life patients with NSCLC and report the clinical factors characterizing this population. Secondary endpoints were rate- and duration of intracranial response  in those with recently treated versus untreated BM (Group A versus Group B). In addition, level of concordance between ICR and extra cranial response (ECR) was addressed. Furthermore, OS, QoL (change at 8–9 weeks compared to baseline) and PFS of patients without BM compared to patients with BM.Relevant patient demographics were registered at the time of inclusion in a REDcap database . ComorbiIn order to avoid delay of ICI initiation, baseline MR-C was allowed on day −30 to +7 in relation to 1st ICI. The MR-C included pre- and post-contrast T1-weighted, T2 weighted and/or T2-Fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted imaging (DWI) sequences in line with the current recommendation . BaselinResponse evaluation of BM was based on modified RECIST 1.1 (mRECIST) criteria allowing target lesions in the CNS of ≥5 mm (or at least twice the MR slice thickness) . In caseEvaluation of QoL was performed at baseline within 1 week prior to first ICI and at week 8–9 using the validated 5-level EQ-5D version produced by the EuroQol group (EQ-5D-5L) . This ist-test or Wilcoxon’s signed rank test for continuous variables when applicable. OS and PFS were examined using survival curves based on Kaplan–Meier (KM) estimates. Inverse KM was used to estimate median follow-up time. A Cox proportional hazards regression model was used to evaluate factors associated with OS in addition to the log-rank test. Based on univariate Cox regression analysis, a multivariate Cox regression model was constructed, including known outcome predictors such as PD-L1 expression and performance status as well as factors from the univariate analysis with a p-value of <0.2. Checking the proportional hazard assumption was performed using Schoenfeld residuals. For comparison of survival of patients with no BM, untreated BM and all prior treated BM, the log-rank test for trend was applied. Change from baseline to week 9 in both EQ-index-score and in EQ-VAS  was captured using a paired t-test. The index score was calculated only if patients had completed all five questions. Only patients who had completed both baseline QoL and QoL at week 8–9 were included in the analyses of EQ-VAS and EQ-index-score. Statistical analysis was performed using STATA version 17.0 with two-sided tests and a level of p < 0.05 was considered statistically significant [Baseline characteristics were compared using the chi-square or Fischer’s exact test for categorical variables and the unpaired nificant .ClinicalTrials.gov (identifier: NC T03870464). Data were deposited in a REDcap database at Odense patient explorative network (OPEN) with project number OP-521.The study was approved by the regional research ethical committee of Southern Denmark (Project-ID: 20170155) and was conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent prior to participation. The study was registered at Patients were enrolled from 1 April 2018 through 31 April 2021 at the Department of Oncology, Odense University Hospital Denmark. Data cut-off was 7 September 2021.  or CT-C (n = 5) obtained. Median follow-up time was 23.2 months (IQR 16.4–30.2). Two patients had EGFR mutation (2/133) and none had ALK-fusion oncogene (0/133). A total of 45 patients (28%) had BM at baseline, with the majority (n = 32) being identified due to screening. The vast majority had active BM (n = 40) compared to stable BM (n = 5). Neurologic symptoms were described in 18 patients with headache being the most frequent. Patients with BM were younger, more often had a histology of adenocarcinoma, and more often used CS both prior to and during the ICI course than those without BM (n = 5) or increase in clinical symptoms (n = 4)  . Four paFor data on all patients with BM, see A total of 85 out of 159 patients had died at time of follow-up. No statistically significant difference in OS in patients with baseline BM compared to patients without was seen, as illustrated in The latter illustrates OS in all patients with previously treated compared to untreated BM and those without BM. For group A, mOS was 12.3 months (95% CI: 5.2-NR) versus 20.5 (95% CI: 4.9-NR) in group B . For data on PFS, rICR, ECR and dICR, see Of the 159 patients, 151 (95%) completed baseline QoL assessment and 106 (67%) at week 8–9 . A complp < 0.019 and a mean EQ-index-score improvement from 0.73 (95% 0.69–0.77) to 0.80 (95% 0.77–0.84), p < 0.001. No statistically significant improvement was obtained for patients with BM, with mean EQ-VAS changing from 72.8 (95% CI 64.7–80.8) compared to 76.3 (95% 67.7–84.8), p = 0.313 (p = 0.56.Patients had a mean EQ-VAS improvement from 65.2 (95% CI 60.4–70.0) to 70.7 (95% CI 88.6–74.7),  = 0.313  and an EOf those with BM, Group B had the highest baseline QoL at week 8–9 (EQ-VAS at week 8–9 was 87.5),  and the To the best of our knowledge, this is the first real-life prospective study on prevalence of BM in ICI eligible patients with NSCLC providing data on QoL and intracranial response to ICI in relation to long-term OS. We reported that 28% of real-life patients with NSCLC currently eligible for ICI had BM, which was higher than the 6–17% with BM participating in the pivotal RCTs ,17,30,31Current data on patients with NSCLC and BM receiving ICI come from RCTs, single arm phase I/II trials, or expanded access programs, all of which have included pre-selected patients, resulting in almost no data on patients with active and/or symptomatic BM ,35,36. TImportantly, we report that patients with BM have a mOS, mPFS and baseline QoL level fully comparable to patients with NSCLC without BM. Patients with BM did, however, more often use CS both prior to and at the time of ICI initiation, which might reflect that these patients have different symptoms. This might need attention. The improvement in QoL at week 8–9 was only statistically significant for patients without BM, illustrating the overall early benefit of ICI in this patient group. In multivariate analysis, the well-established biomarker PD-L1 ≥50% was a predictor of better outcome, in line with the present literature. Bone metastasis, on the other hand, was only one of several metastatic sites in this study that significantly impaired OS. Most RCTs including ICI do not include baseline data on bone metastasis, but two recent nationwide Danish retrospective studies reported that bone metastasis as opposed to brain metastasis may be considered an important negative predictor of poorer survival ,41. OtheDespite the lack of consecutive MR-C in those patients who did not present with BM at baseline, we reported that 11% 13/114) of those without baseline BM developed symptomatic BM verified by MR-C later on during their disease course (not necessarily during ICI). This number added to the 28% having baseline BM , is in alignment with the literature in general, stating that 30–50% /114 of t. This isSince our study illustrates that patients with NSCLC and BM might obtain durable responses leading to long-term survival without early impairment of QoL, it is important that these patients are offered ICI. Before the implementation of ICI for patients with NSCLC, long-term survival in patients with BM was rare except for those with driver mutations/alterations . ImportaThe limitations of this study are, among others, the small size of the study population and the lack of randomization based on the distribution of local therapy or not. By performing descriptive and exploratory comparisons among group A and B, we tried to gather clinically important and warranted data. A limitation was that these groups were not matched in terms of known prognostic factors. Probably due to clinicians’ selection bias, patients in the untreated group were more often treated with ICI in first line, and both size and number of lesions were smaller/lower. This probably also resulted in a higher baseline QoL. Data on the investigated subgroups are presented in The comparable OS in patients with BM compared to those without BM was in correlation with the few previous studies from malignant melanoma ,34 and tA recent RCT in patients with malignant melanoma did include patients with symptomatic BM using combination ICI therapy and reported benefits even in this subgroup, but to a lesser extent than those who were asymptomatic . As in tThis study illustrates that patients with advanced NSCLC and BM receiving palliative ICI have a long-term survival comparable to those without BM. MR-C screening for BM in patients with advanced NSCLC prior to palliative ICI should be considered standard in order to optimize each patient’s individual treatment course. Since intracranial response was observed in 50% of patients with locally untreated BM with a high correlation to extracranial response, initiation of ICI with close MR-C control but without early local therapy may be an option for selected subgroups with BM. Our data emphasize the importance of including patients with NSCLC and BM in RCTs, and we need large prospective studies investigating combinations of RT and ICI in real-life patients in the nearest future. This is necessary in order to gain more knowledge on how to personalize treatment for this heterogenic group of patients, thereby aiming for further improvement of OS and QoL.


Objective: To explore the role of glycolysis in cardiac fibroblast (CF) activation and cardiac fibrosis after myocardial infarction (MI).Method:In vivo: 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, was injected into the abdominal cavity of the MI or sham mice every day. On the 28th day, cardiac function was measured by ultrasonic cardiography, and the hearts were harvested. Masson staining and immunofluorescence (IF) were used to evaluate the fibrosis area, and western blot was used to identify the glycolytic level. In vitro, we isolated the CF from the sham, MI and MI with 2-DG treatment mice, and we also activated normal CF with transforming growth factor-β1 (TGF-β1) and block glycolysis with 2-DG. We then detected the glycolytic proteins, fibrotic proteins, and the concentrations of lactate and glucose in the culture medium. At last, we further detected the fibrotic and glycolytic markers in human fibrotic and non-fibrotic heart tissues with masson staining, IF and western blot.Result: More collagen and glycolytic protein expressions were observed in the MI mice hearts. The mortality increased when mice were treated with 2-DG (100 mg/kg/d) after the MI surgery . When the dosage of 2-DG declined to 50 mg/kg/d, and the treatment was started on the 4th day after MI, no statistical difference of mortality between the two groups was observed . The collagen volume fraction was smaller and the fluorescence signal of α-smooth muscle actin (α-SMA) was weaker in mice treated with 2-DG than PBS. In vitro, 2-DG could significantly inhibit the increased expression of both the glycolytic and fibrotic proteins in the activated CF.Conclusion: Cardiac fibrosis is along with the enhancement of CF activation and glycolysis. Glycolysis inhibition can alleviate cardiac fibroblast activation and cardiac fibrosis after myocardial infarction. Heart failure is a cardiovascular disease with high morbidity and mortality, which cause a great burden on society . Among tvia several ways in fibrotic diseases. It not only produces several key metabolites responsible for CF activation, like glycine and triphosadenine, it also produced abundant lactate, which was important for the activity of proline hydroxylase, TGF-β1 and the hydroxylation of collagen , we demonstrated the role of glycolysis in the fibrotic process of the heart. At last, we also measured the glycolysis change in human fibrotic and non-fibrotic tissues, thus providing more clinical evidence.C57BL/6J mice were used to perform a MI or sham surgery. Then 2-DG  was delivered by intraperitoneal injection immediately after the surgery or started at the 4th-day after the surgery. Trans-thoracic echocardiography was performed on the 28th day after the surgery to evaluate the cardiac function. The animal use protocol was approved by Institutional Animal Care and Use Committee, Sun Yat-sen University. Detailed information was provided in the Human heart specimens were harvested during cardiac surgeries in Sun Yat-sen Memorial Hospital. Among them, the resected ventricular aneurysm tissues were used as fibrotic tissues while papillary muscle tissues from the diseased valve were served as non-fibrotic tissues. The study design was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital.Heart tissues from the mice or patients were collected and fixed in 4% paraformaldehyde in phosphate-buffered saline  overnight after being perfused with cold normal saline. Then the hearts were processed for paraffin embedding and subsequently cut into slices. Slices then undergo dewaxing, rehydration and stained for collagen fibers with Masson's Trichrome staining Kit  following the manufacturer's instructions. Fibrotic tissues and muscle tissues were segmented using ImageJ (NIH) and fibrosis was expressed as the percentage of fibrotic tissue in each section.Slices from human or mice undergo dewaxing, rehydration, antigen retrieval and blocking. Then tissues were incubated with primary antibodies overnight, followed by fluorescigenic secondary antibodies incubation. At last, the nucleus was stained, and photos were taken with the fluorescence microscope. The fluorescence signal quantification was conducted with Image J.in vitro experiments. The NMCFs were separated from the ventricle of the neonatal 1–3-day-old mice as described previously (https://www.sciencellonline.com/human-cardiac-fibroblasts-juvenile-ventricular.html).Neonatal mouse CFs (NMCFs) and human CFs were used for the eviously , and theHuman CFs at the passage between 6 and 8 and NMCFs at passage 2–3 were pre-treated with 2-DG (1 mmol/L) for 1 h. Then human/mouse transforming growth factor-β1  was added to induce an activated phenotype. Cells were treated for 48 h and harvested. For HK2 knockdown tests, NMCFs were treated with small interfering RNA against HK2 (si-HK2: GGACAAGCUACAGAUCAAAdTdT) or scrambled siRNA . Cell medium was refreshed after 12 h treatment and harvested after 48 h cultivation.Adult mouse CFs (AMCFs) were separated from the ventricle of the MI or sham mice with or without 2-DG treatment on the 28th day after the sham or MI surgery as described previously , 21. As Lactate in the cell medium were detected with Lactate Assay Kit II , and the glucose in the cell medium were detected with Glucose Colorimetric/Fluorometric Assay Kit  according to the manufacturer's instructions. All results were normalized to the total protein concentration.Western blot was performed as previously described . AntibodCells were fixed with 4% paraformaldehyde . After being permeabilized with 0.5% Triton X-100  for 20 min, cells were blocked with 5% bovine serum albumin . Afterwards, cells were incubated with the indicated the primary antibodies overnight and then the fluorescigenic secondary antibodies for 1 h at room temperature. At last, fluorescence were captured with the fluorescence microscope. Antibodies are listed in Bonferroni test. Student's t-test was used to analyze differences between two groups. Kaplan-Meier method was used for the survival analysis, and the log-rank test was used for the statistical analyses. P-value < 0.05 indicated statistical significance.Data were showed as mean ± SEM. Statistical analyses were performed with GraphPad Prism Software (version 8.0.1). Statistical comparison among multiple groups was carried out by one-way ANOVA followed by the vs. MI: LVEF (%): 75.84 ± 0.96 vs. 21.46 ± 3.70, LVFS (%): 43.67 ± 0.80 vs. 9.87 ± 1.83, LVDd (mm): 3.44 ± 0.10 vs. 4.54 ± 0.38, LVSd (mm): 1.94 ± 0.07 vs. 5.04 ± 0.41; P < 0.05). Masson staining showed that plentiful collagen was deposited in the heart after MI (collagen volume fraction (%): sham vs. myocardial infarction: 1.93 ± 0.71% vs. 26.49 ± 4.87%; P < 0.05; To explore the role of glycolysis in cardiac fibrosis, we measured the glycolytic markers in the fibrotic heart after MI. As is shown in P < 0.05, P = 0.98, P < 0.05; Next, we aimed to figure out whether cardiac fibrosis could be reversed when glycolysis was inhibited. We delivered a glycolysis inhibitor, 2-DG (100 mg/kg/d) or an equal volume of PBS into mice in the MI group by intraperitoneal injection immediately after the surgery and maintained the injection daily. We found that the mortality increased in MI mice with 2-DG injection than that with PBS injection . To further ensure that glycolysis inhibition could alleviate the CF activation in vivo, we isolated the AMCFs from the MI or sham mice with or without 2-DG treatments on the 28th day after the surgery. As is shown in in vitro test to make sure whether 2-DG could inhibit CF activation directly. We used TGF-β1 to induce human cardiac fibroblast activation. In CF activation was the main force for cardiac fibrosis after MI. Thus, we then detected the markers of CF activation (α-SMA) in the heart tissues of mice in sham, MI, and MI combined 2-DG groups. As is shown in in vivo and in vitro.Consequently, our results demonstrated that glycolysis inhibition could reverse the fibroblast activation vs. fibrotic heart tissues: 17.48 ± 1.84 vs. 82.45 ± 10.45, P < 0.05; vs. fibrotic heart tissues: 0.38 ± 0.11 vs. 2.48 ± 0.74, P < 0.05; To acquire the clinical evidence, we further detected the fibrotic and glycolytic markers in human fibrotic and non-fibrotic heart tissues. The clinical information of enrolled patients was shown in Although treatments for heart failure have made great progress, it is still a global refractory disease with high mortality and rehospitalization rate . RegardlIn our study, glycolysis inhibition could alleviate the CF activation and cardiac fibrosis after MI, just as the previous reports in other fibrotic diseases , 23, 24.In our study, 2-DG delivery from the day of MI surgery at a dosage of 100 mg/kg/day increased the mortality of mice. After we lowered the dosage of 2-DG to 50 mg/kg/day and delayed the time of initial drug administration to the 4th day after the surgery, the mortality of the mice decreased, and the fibrosis area of the heart lessened. To our knowledge, fibrosis is an important event and a dynamic process after MI . In the In our study, we did not explore the further mechanism of the anti-fibrotic effect of 2-DG. 2-DG might contribute to CF activation in several ways. As several critical metabolites in glycolysis were responsible for CF activation, like glycine and triphosadenine , 2-DG miin vivo and in vitro, we cannot eliminate the possible confounding contribution by other cardiac cells. Secondly, we explored the role of glycolysis in cardiac fibrosis and CF activation after MI. We did not further explore the role of tricarboxylic acid cycle and oxidative phosphorylation. Thirdly, as human tissues were hard to acquire, especially normal heart tissues and tissues from remote sections of the human MI heart, we had to use papillary muscles tissues as candidates of non-fibrotic heart tissues.Some other limitations should be acknowledged in our study. Firstly, we delivered 2-DG by intraperitoneal injection, which may exert a systemic effect. Though we have demonstrated the role of glycolysis on CF activation In conclusion, our study demonstrates that glycolysis inhibition can alleviate CF activation and cardiac fibrosis after MI. Glycolysis may be a new target for the treatment of cardiac fibrosis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Sun Yat-sen Memorial Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Institutional Animal Care and Use Committee, Sun Yat-sen University.Z-TC, Q-YG, and M-XW were responsible for most of the experiments. MW provided the human heart specimens. YJ, R-LS, and D-CG provided the experimental assistances. QG, C-YL, XL, and S-XC provided writing assistances. J-FW, H-FZ, and Y-XC designed and directed the study. All authors have read and approved the final submitted manuscript.This work was supported by grants from the National Natural Science Foundation of China , Guangdong Basic and Applied Basic Research Foundation , the Yat-sen Start-up Foundation (No. YXQH202014), the Science and Technology Program of Guangzhou City of China (201803040010), the Guangzhou Regenerative Medicine and Health Guangdong Laboratory (No. 2019GZR110406004), and the Guangzhou Key Laboratory of Molecular Mechanism and Translation in Major Cardiovascular Disease (No. 202102010007).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In conclusion: serum IL-13, Vitamin D, and miRNA-135a could be potential biomarkers in monitoring DAA treatment and HCC prediction. DAAs-induced SVR may decrease the incidence of HCC.Direct-acting antivirals (DAAs) are used for hepatitis C virus (HCV) treatment. However, treatment failure and hepatocellular carcinoma (HCC) development following treatment was reported. In this study, we assessed the role of serum vitamin D, interleukin 13 (IL-13), and microRNA-135a in the prediction of treatment failure with DAA and HCC development among Egyptian HCV-infected patients. A total of 950 patients with HCV-related chronic liver disease underwent DAA treatment. Before DAAs, serum vitamin D and IL-13 were determined by ELISA, and gene expression of miRNA-135a was assessed in serum by real-time PCR. The predictive abilities of these markers were determined using the receiver operating characteristic (ROC) curve. Sustained virological response (SVR) was achieved in 92.6% of HCV-infected patients (responders). High viral load, IL-13, miRNA-135a, and low vitamin D levels were associated with treatment failure and HCC development. HCC development was recorded in non-responders, but not in the responders (35.7% vs. 0%  Chronic hepatitis C (CHC) infection is a global disease that has a variable course. It is a main cause of liver cancer and cirrhosis ,2. HowevVitamin D (vit D) has anti-inflammatory and anti-fibrotic functions that can affect the treatment outcome and minimize HCV-mediated liver disease ,16,17. PInterleukin-13 (IL-13) is a glycosylated polypeptide of the family of IL-13/IL-4 ,25. IL-1miRNA-135a is elevated broadly across many cancer types and a candidate driver of HCV-associated HCC . miRNA-1HCC is a global health dilemma. Primary liver cancer is the seventh most-prevalent cancer that can lead to a high mortality rate ,38. The In this study, we evaluated the association between the selected biomarkers: Vit D, IL-13, and miRNA-135a, and treatment outcome of HCV with DAAs and the risk of development of HCC in Egyptian HCV-infected patients who received DAA therapy in the form of “sofosbuvir and daclatasvir ± ribavirin”. The previous regimen is an acceptable protocol provided by the Egyptian Ministry of Health through the National Committee for Control of Viral Hepatitis (NCCVH) and showed efficacy over 90% .This study was carried out at the Medical Research center, Faculty of Medicine, Assiut University, Egypt from December 2018 to November 2020. This work has been approved by the Local Ethical Committee of the Faculty of Medicine, Assiut University (IRB no. 17300237 and 17300672). All participants were adults and all of them provided written informed consent before collection of samples.A total of 950 patients with treatment-naïve HCV-related chronic liver disease in addition to 50 healthy controls were enrolled in the study. Patients were selected from those attending the AL-Rajhi Liver Center, Assiut University Hospital, Egypt, to dispense DAA treatment during the 100 million healthy lives campaign. AL-Rajhi Liver Center is a large referral tertiary center that provides surveillance, counselling, investigation, and treatment for HCV-infected patients in governorates of Upper Egypt, and it is one of the hospitals that participated in the campaign. Eligible patients were diagnosed with CHC infection based on the presence of anti-HCV antibody levels and detectable serum HCV RNA. The enrolled patients were either non-cirrhotic or cirrhotic. Cirrhosis was determined by fibrosis-4 (FIB-4) score with a FIB-4 >3.25 for advanced fibrosis/cirrhosis and transient elastography  with a liver stiffness ≥14.6 kPa .n = 50) were negative to HBV and HCV markers, and they had no hepatic diseases and were sex- and age-matched with patients. They were selected randomly from outpatient clinic and relatives of the patients. We excluded patients treated with interferon-based regimens or immunomodulating agents. Additionally, patients who were co-infected with hepatitis A and B viruses or human immunodeficiency viruses, those having alcohol or drug-induced liver diseases, those with positive anti-schistosomal antibodies, and those with evidence of HCC before DAA therapy were excluded from this study. The healthy controls  and daclatasvir (60 mg) ± ribavirin  available at the time of enrolment . The comBlood samples were collected into plain tubes labeled with the patient’s name, sex, age, and the date of collection. Serum was isolated from blood and divided into two tubes; the first tube was used for determination of serum level of vit D and IL-13 and the second tube was used for RNA extraction to be used in quantitative real-time PCR reactions (RT-qPCR) for estimating the level of miRNA-135a gene expression.Total RNA was isolated from serum samples using miRNA isolation Kit  following the manufacture’s instruction. cDNA was synthesized using Universal cDNA Synthesis Kit . 10 µL of reverse transcription reactions was used for Syber green based miRCURY LNA Universal RT microRNA PCR assay. U6 was used as an internal reference. The primers for miR-135a and U6 were as follows: miR-135a ; U6 . PCR conditions for miR-135a amplification were 95 °C for 20 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 20 s, with a final incubation at 70 °C for 5 s. qRT-PCR was carried out under optimal conditions. Expression level of miR-135a was normalized to U6 and determined using the 2-DDCt method .Vitamin D was determined in serum using Human Vitamin D Promokine (direct) ELISA kit  according to the manufacturer’s instructions. Briefly, 20 µL of standards/controls/serum was added into the wells. Strips were incubated for 45 min at room temperature on horizontal shaker, then 150 µL of anti-vitamin D antibody (AB) was added into each well. The plate was covered and incubated for 45 min at room temperature on a horizontal shaker. After that, the content of each well was discarded, and plate was washed 5 times using 250 µL of wash buffer. After the final washing step, 200 μL of conjugated CONJ was added into each well, and the plate was incubated for 45 min at room temperature on a horizontal shaker. The washing steps were repeated again and then 200 µL of substrate was added into each well and incubated for 10–15 min at room temperature in the dark. The reaction was stopped by addition of 50 µL stop solution into each well. Absorption was determined immediately with an ELISA reader at λ 450 nm. ® Human IL-13 ELISA Kit  according to the manufacturer’s instructions Briefly, 100 µL of each standard and sample was added into appropriate wells, then the wells were incubated for 2.5 h at room temperature with gentle shaking. After the washing step. A total of 100 µL of 1X prepared biotinylated antibody was added to each well and incubated for 1 h at room temperature with gentle shaking. Then, the plate was washed and 100 µL of prepared Streptavidin solution was added to each well and incubated for 45 min at room temperature with gentle shaking. After the washing step, 100 µL of TMB one-step substrate reagent was added to each well and incubated for 30 min at room temperature in the dark with gentle shaking. After the color development, 50 µL of stop solution was added to each well, and the plate was read using ELISA reader at λ 450 nm.IL-13 was determined in serum using RayBio® HCV RG RT-PCR Kit  according to the manufacturer’s instructions. Briefly, desired number of PCR tubes were placed into the adapters of the cooling block. The reaction mix typically contains all the components needed for PCR except the sample. 30 µL of the master mix were added into each PCR tube, then 20 µL of the eluted sample RNA were added. Correspondingly, 20 µL of at least one of the quantitation standards (Hep. C Virus RG QS 1–4) must be used as a positive control and 20 µL of water  as a negative control. The PCR tubes were closed. The locking ring (accessory of the Rotor-Gene Instrument) was placed on top of the rotor to prevent accidental opening of the tubes during the run. For the detection of HCV RNA, a temperature profile was created according to manufacturer’s instructions.HCV-RNA was determined in serum using artust-test, ANOVA or Mann–Whitney U-test or Kruskal–Wallis test for two or more groups of normally or abnormally distributed data, respectively. Qualitative data were expressed as a percentage and compared using chi-squared (χ2) or Fisher’s exact probability test. The receiver operating characteristic curves (ROC) were plotted to measure the performance of investigating parameters in predicting the treatment failure and HCC and to select its optimal cut-off value at which the sensitivity, specificity, and positive likelihood ratio. All tests were two-tailed and p-values < 0.05 were considered significant.All statistical analyses were done by using GraphPad Prism 8.4.0, Statistical Package for the Social Sciences (SPSS) for Windows version 16  and MedCalc program. The Kolmogorov–Smirnov test of normality was used to test the normality of data. The quantitative data were expressed as mean ± standard deviation (SD) or median and the range (minimum–maximum) for normally or abnormally distributed data, respectively. They were compared using Student’s 5 IU/mL) at the start of therapy compared with non-responders (9.6 × 105 IU/mL). None of the responders had developed HCC during the follow-up period, while 35.7% (25/70) of the non-responders had developed HCC.A total of 950 patients with HCV-related chronic liver disease  were included in this analysis. The patients received DAA therapy (sofosbuvir and daclatasvir ± ribavirin” for 12 weeks), and they were followed up 6 months after the end of therapy to monitor the therapy outcome. Out of 950 patients, 880 patients (92.6%) achieved SVR (responders), and HCV RNA was not detectable in the serum of these patients, while 70 patients (7.36%) did not achieve the SVR (non-responders). We assessed the demographic and clinical characteristics of responders and non-responders to identify the risk factors associated with the therapy failure. There was no significant difference between responders and non-responders in terms of age, sex, and smoking , while wThen, we assessed the levels of these parameters  in responders and non-responders in both cirrhotic and non-cirrhotic patients . In cirrOut of 70 non-responders, 25 patients (35.8%) had developed HCC following DAA therapy, while 45 patients (64.2%) had not developed HCC. We assessed the demographic, and clinical characteristics of non-responders to identify the risk factors and predictive markers for HCC development following the therapy failure. We found that age, sex, smoking, and comorbidities such as diabetes and hypertension were not significantly different between non-responders who developed HCC and non-responders without HCC , while tWe assessed the diagnostic utility of IL-13, miRNA-135a, and vit D to predict the therapy failure and the development of HCC. To this end, we generated the receiver operating characteristic curve (ROC) that plots sensitivity versus 1-specificity to evaluate the overall performance of each marker. Using the serum levels of IL-13 as a cutoff to discriminate between responders and non-responders, the AUC was 0.999 (95% CI (0.998–1)) and serum IL-13 > 135.9 pg/mL had a sensitivity of 100% and specificity of 96% to differentiate between responders and non-responders a. The AUThen, we assessed the possibility of using these markers to predict the development of HCC. ROC analysis revealed that the AUC was 0.992 , 0.973 , and 0.872 (0. 809–0.935) for IL-13, miRNA135a, and vit D, respectively. A serum level of IL-13 of >249 pg/mL can differentiate between the non-responders who developed HCC and HCV patients who did not develop HCC with a 100% sensitivity and 98.4% specificity. Similarly, the cut-off value of serum miRNA135a >3.66-fold change can discriminate between HCC and non-HCC with 100% sensitivity and 89% specificity. Finally, the serum level of vit D <21.04 ng/mL can differentiate the HCC patients from non-HCC patients with an 80% specificity and 92% sensitivity b. AdditiHCV infection is one of the major health problems worldwide. Egypt has the highest prevalence of HCV infection ,59. PrevChronic HCV-infected patients who received DAAs were divided into two groups: responders and non-responders, according to the SVR. The non-responders were divided based on the complications associated with therapy failure into patients who developed HCC and patients without HCC. Our results revealed that diabetes, hypertension, and liver cirrhosis were risk factors for therapy failure. Moreover, the HCV load, elevated serum level of IL-13, and upregulation of miRNA-135a expression was linked to the unresponsiveness to DAA therapy and persistent infection. Similarly, several studies reported the presence of a significant link between diabetes, hypertension, and viral load with HCV therapy failure ,75,76,77Additionally, we showed that a lower serum level of vit D in chronic HCV-infected patients was associated with therapy failure and the development of HCC. In a parallel line, Abd Allah et al. reported that CHC is associated with vitamin D deficiency but iron overload, resulting in a reduced level of hepcidin. Treatment with sofosbuvir/daclatasvir increases hepcidin, thereby reducing iron levels and increasing vit D . A studyIn addition, we evaluated the risk factors and prognostic markers for the acquisition of HCC after DAA treatment. We found that high viral load, elevated IL-13 and miRNA135a levels, and reduced serum levels of vit D were associated with HCC development following the DAA failure. HCV infection can progress to cirrhosis-related HCC . SeveralInterestingly, high HCV load was a risk factor for therapy failure and the development of HCC in non-responders. Similarly, Noh et al. reported that serum HCV viral load is a risk factor for the development of HCC but not liver-related mortality . Higher ROC analyses for serum IL-13, miRNA-135a, and vit D revealed that IL-13 (>135 pg/mL) has better prognostic accuracy for predicting the treatment failure with 100% sensitivity and 96% specificity, followed by miRNA 135a with 97% sensitivity and 91% specificity. In addition, serum IL-13 (>249 pg/mL) can predict the development of HCC with 100% sensitivity and specificity of 98% and 89% for IL-13 and miRNA 135a, respectively. These markers can be diagnostically useful for the clinician to predict the therapy outcomes and the possibility of HCC development, especially in non-responders. Clinicians can benefit from the markers to take the appropriate preventive measures to reduce the complications associated with the treatment failure. It is important to highlight that the level of these markers could differ at other places depending on the patient criteria, cohort size, instruments, and methodology.The manuscript has number of advantages including the recruitment of large number of participants who received HCV-DAA therapy, association between some selected parameters  and HCV treatment outcome and development of HCC. On the other hand, this study also has some limitations. The exact mechanism of resistance to the DAA was not analyzed. We analyzed the changes in IL13 only; however, the modulation of the other pro and anti-inflammatory cytokines was not investigated. In addition, the association between response to therapy and other micro-RNA was not analyzed. Furthermore, these parameters were assessed in patients recruited from one center. Moreover, the enrolled patients received one approved regimen (sofosbuvir and daclatasvir ± ribavirin). Future studies including patients from multi-centers and who received different HCV DAA regimens are needed to ascertain these findings.We reported the prognostic markers of IL-13, miRNA135a, and vit D associated with DAA treatment failure in chronic HCV-infected patients receiving sofosbuvir and daclatasvir ± ribavirin. Additionally, we showed that these markers could predict HCC development following failure in DAA therapy.


Coelastrella rubescens dwells in habitats with excessive solar irradiation; consequently, it must accumulate diverse compounds to protect itself. We characterized the array of photoprotective compounds in C. rubescens. Toward this goal, we exposed the cells to high fluxes of visible light and UV-A and analyzed the ability of hydrophilic and hydrophobic extracts from the cells to absorb radiation. Potential light-screening compounds were profiled by thin layer chromatography and UPLC-MS. Coelastrella accumulated diverse carotenoids that absorbed visible light in the blue–green part of the spectrum and mycosporine-like amino acids (MAA) that absorbed the UV-A. It is the first report on the occurrence of MAA in Coelastrella. Two new MAA, named coelastrin A and coelastrin B, were identified. Transmission electron microscopy revealed the development of hydrophobic subcompartments under the high light and UV-A exposition. We also evaluate and discuss sporopollenin-like compounds in the cell wall and autophagy-like processes as the possible reason for the decrease in sunlight absorption by cells, in addition to inducible sunscreen accumulation. The results suggested that C. rubescens NAMSU R1 accumulates a broad range of valuable photoprotective compounds in response to UV-A and visible light irradiation, which indicates this strain as a potential producer for biotechnology.The microalga  The surfaces of stones, buildings and tree bark can be covered by an orange or red crust. This crust is of a biological nature: it is formed by aeroterrestrial microalgae. These algae are among the most ubiquitous microbes on the aerial surface . AeroterMicroalgae are oxygenic phototrophic organisms. On the one hand, light is vitally important for their existence, because it promotes electron transport in the photosynthetic electron transport chain (ETC), a main source of assimilatory power for the synthesis of organic substances in the cell. On the other hand, it is the cause of photodamage, a set of processes leading to light-driven destructions in the cell. Thus, phototrophic organisms must constantly avoid and dispose of the harmful power of electromagnetic radiation. Being exposed to the sunlight directly falling on the surface of the earth, aeroterrestrial species are especially vulnerable to photodamage ,3,4. In A- and QB-binding sites +, of most natural compounds in the ESI-MS undergo fragmentation in an ionization source. This results in the formation of characteristic fragment ions [m/z = 343.1 of the protonated molecule [M+H]+ in the fraction with Rt = 0.39 min . Saturated mycosporine-glycine:valine has an absorption maximum at 335 nm in 80% methanol [Phaeocystis [C. rubescens NAMSU R1 water–methanol extract exhibited the maximum at a shorter wavelength -mycosporine-glycine:γ-dehydrovaline  that had not previously been reported [Collectively, we found two MAA with a determined chemical formula in reported ,72,73,74C. rubescens might be an interesting model object for the studying of adverse effects on phototrophs. The UV-protective role of MAA has been shown in many studies previously [C. rubescens are known as a UV-tolerant alga [C. rubescens NAMSU R1.From an ecological point of view, MAA are an adaptation to a harmful effect of UV irradiation and other stress factors, such as osmotic or drought stress. In the case of organisms inhabiting the deep of seas and oceans, the task of photoprotection is partially solved, due to shielding beneath the surface water layers. By contrast, phototrophs in the upper water layers with high irradiation (photic zone), as well as aeroterrestrial species, should have more effective photoprotective mechanisms ,4. Indeeeviously ,13,14. Teviously ,13,17,24eviously ,16,17,24ant alga . At the ant alga . We haveC. rubescens NAMSU R1 cells after HL and HL+UV-A treatment indicated that their synthesis in the microalga was HL- and UV-A-inducible. It could also be concluded that visible light and UV-A had a synergetic effect in this microalga, because a higher absorbance increase was observed in the case of HL+UV-A treatment, rather than in the case of HL only. This was in accordance with previous observations, that both high light and UV-A induce MAA accumulation [Agarophyton tenuistipitatum [Aphanothece [Chlorella, Stichococcus, and Pseudococcomyxa [Porphyra columbina shows a decrease in MAA levels under HL+UV-A+UV-B, rather than under HL+UV-A only [Lobosphaera incisa SAG 2007 [Mazzaella laminarioides, treatment by both UV-A and UVA+UV-B leads to a reduction in MAA content [C. rubescens could be a matter of further works. MAA synthesis might be mediated by photoreceptors, e.g., phytochrome A, cryptochromes and phototropins. Their chromophore groups absorb UV-A radiation [The increase in the absorbance of the characteristic band of MAA in mulation . There imulation ,14,24. Foccomyxa . At the adiation ,11. NeveC. rubescens NAMSU R1 cell suspensions that compensated to light scattering were characterized by a strong band at 250–350 nm  exhibit a wide range of protective mechanisms against sunlight. They include ROS-neutralizing enzymes, cycles of carotenoid oxydation/de-epoxidation and DNA reparation ,12,13,32C. rubescens NAMSU R1 are shown in C. rubescens NAMSU R1 treated by HL or HL+UV-A demonstrated lower values of the PS II maximal photochemical quantum yield (Fv/Fm) than the cells treated by LL  [The representative chlorophyll induction curves of ed by LL a. Such aacustris ,40,81,82. salina . This co. salina ,84,85,86ed cells b. A shar source) . It was Absorption of PAR and UV by photosynthetic pigments causes the destruction of intracellular structures. The damaged chloroplast is a source of ROS and is not able to neutralize their high amount ,86. ThusC. rubescens NAMSU R1, after their irradiation. They were similar to the epichloroplast membrane structures (EMS), described by Gorelova et al. [C. rubescens cells pointed to the emergency evacuation of the thylakoid membranes, and the chloroplast envelope was caused by the harsh effects of irradiation. The matter formed after the disassembly of PSA and other chloroplast components, with the resulting autophagy most likely being intended to fill in part the pool of neutral lipids [That mechanism is conditioned by the rearrangement of membrane structures in the cell PSA and could be considered an autophagy-like process. In that case, thylakoids and sometimes whole chloroplasts are degraded to decrease the ability of cells to absorb light energy, which decreases the risk of photooxidative damage under stress conditions ,85,88. Ua et al. . HL-treaa et al.  the trana et al. . The lasa et al. ,91,92. Tl lipids ,94,95. Fl lipids ,93. The H. lacustris, the reduction in PSA is important for the viability of cells under light, after their freezing [H. lacustris leads to an increase in carotenoid accumulation. This indicates competition between these photoprotective mechanisms [Chromochloris zofingiensis [Being the main site of photodamage, photosynthetic membranes should be reduced under HL, in order to avoid the irreversible destruction of the cell. In carotenoid-accumulating microalgae, the disassembly of PSA plays an important role in photoprotection along with carotenoid accumulation. Indeed, in freezing . The inhchanisms . The samngiensis . At the Malus × domestica bark in the Rastorguevo Village, Moskovskaya oblast, Russia (55.55 N 37.70 E), in July of 2020. Strain isolation and maintenance were performed, based on previously developed protocol for the isolation of green carotenogenic microalgae [The strain NAMSU R1 was isolated from the dry reddish crust a on the croalgae .2/s at 24 °C at 85 r.p.m in the New Brunswick Innova 44 shaking incubator .To estimate the algal biomass accumulation, the strain NAMSU R1 was cultivated in 500 mL of the BG-11 media  in two 1Primary identification and detailed morphological characterization and ultrastructure were performed by the evaluation of the culture by light and electron microscopy.Pectinodesmus pectinatus CCAP 276/40 (Scenedesmaceae) was taken as an outgroup. Data analysis for the study of phylogenetic relationships was performed in MEGA X [A precise identification was based on the sequence of ITS1-5.8S rRNA-ITS2. DNA extraction, fragment amplification and sequencing were performed, as previously described . Sequencn MEGA X . Sequencn MEGA X . Phylogen MEGA X . The Kimn MEGA X  with an n MEGA X  and the n MEGA X , with 102) and a cold-white LED , and of 150 μmol/m2/s . Photon flux density of the visible light was measured at the level of cell suspensions by a LI-COR LI-250A quantum meter, with a cosine-corrected sensor .The following LEDs were used in the work: UV-A LED . The cells were transferred to 50 mL of BG-11 medium and diluted to the final optical density at 660 nm (D660) of 0.51 units. The strain NAMSU R1 was cultured in the 250 mL T-75 TC-treated cell culture flasks  at 25 °C for three weeks. Two experimental variants were considered: cells were illuminated by HL (HL-variant) and by HL and UV-A (HL+UV-A). The variant of the cells illuminated by LL only was used as a control. The experiments were performed in three replicates.Suspensions of Microalgal cultures were evaluated by bright-field and fluorescent microscopy. This was performed using a Leica DM2500 microscope , equipped with the attached Leica DFC 700T camera. Chlorophyll autofluorescence was excited by a UV-lamp HXP 120 of the same manufacturer, using the band-pass filter Y3 (565–610 nm). The fluorescence was excited by a UV-shutter.v/v) glutaraldehyde and dehydrated through the graded ethanol series. Then, they were transferred to anhydrous acetone and dried at the CO2 critical point in a dryer HCP-2 . The samples then were sputter-coated with gold-palladium in an IB Ion Coater  and examined with a JSM-6380LA  scanning electron microscope at an accelerating voltage of 15 kV.For scanning electron microscopy (SEM) the algal cells were fixed in 2% (v/v) glutaraldehyde solution in 0.1 M sodium cacodylate buffer, at room temperature for 0.5 h, and then post-fixed for 4 h in 1% (wt/v) osmium tetroxide in the same buffer. The samples were embedded in the epoxy resin araldite . Ultrathin cross-sections were made using an ultramicrotome Leica EM UC7  and a diamond knife Ultra 45° . They were placed on a formvar-coated electron microscopy grids and contrasted with lead citrate [For transmission electron microscopy (TEM), the samples were fixed and dehydrated following the standard protocol described in Gorelova et al. : fixed i citrate . Cross-sAbsorbance spectra were registered in the range of 250–800 nm on an Agilent Cary 300  spectrophotometer with a 150 mm integrative sphere CA30I  in 1 cm quartz cuvettes. The measured optical density compensated for the interferences from the incomplete collection of scattered light, as previously described ,108.Aλ, was calculated as:λ of the sample placed at a certain distance apart from the integrative sphere and as close as possible to it, respectively, The absorbance corrected to the effect of light scattering, g. The supernatant was removed, the biomass was frozen at the temperature of N2 boiling, and then the cells were disrupted using a ceramic mortar and a pestle.Putative sunscreens were extracted from the cells according to Folch et al. . Before C. rubescens NAMSU R1 cells as a blank. The carotenoid content in the biomass was determined as described in [Both water–methanol (hydrophilic) and chloroform (hydrophobic) fractions were collected and analyzed. The spectra of the fractions were recorded in the range of 250–800 nm on an Agilent Cary 300  spectrophotometer in 1 cm quartz cuvettes. For HL and HL+UV-A-treated cells, the spectra were registered against the water–methanol extracts of the LL-treated a fluorescence. To compensate for the effect of fluorescence reabsorption due to the high density of cell suspension, the spectra were registered in a series of dilutions. The dilution range characterized by the linear correlation of the dilution and fluorescence intensity was eventually selected for the analysis . Cell suspensions (2 mL) were transferred to a 24-well polypropylene plate . Fluorescence was excited by the light in the UV and visible range of the spectrum ; emission was detected at 750 nm (bandwidth 20 nm)—the band of chlorophyll analysis .f) was calculated for the pigment fractions obtained after separation.The chloroform fraction of cell extracts see  was analv/v) HCOOH in H2O (component A) and 0.1% HCOOH in CH3OH (component B). Elution profile: 1% (v/v) A in B (0–1 min), 1→5% (v/v) A in B (1–5 min). The MS analysis was done in positive-mode ESI MS: m/z in the range of 100–2000, the ion source temperature of 150 °C, the desolvatation temperature of 200 °C, the capillary voltage of 4.5 kV, the ESI voltage of 30 V, and the desalvation gas (N2) flow rate of 461 L/h. Data were analyzed in a MassLynx software .Analysis of the sunscreens from the hydrophilic (methanol–chloroform) fraction see  was perfStationary chlorophyll fluorescence induction curves for the assessment of non-photochemical quenching of the excited chlorophyll states were recorded in a quartz cell (2 mm pathlength) with Fluorpen FP100 s PAM-fluorimeter  after 15 mins dark adaptation, according to previously reported protocol . The foled state  was calcC. rubescens NAMSU R1. A complex of protective mechanisms, including shielding by chemical substances, and autophagy acting through a partial chloroplast reduction, were shown.A sunscreen effect provided by secondary carotenoids and MAA in response to HL and UV-A was described using a new strain of aeroterrestic microalga


Characterization of the naturally acquired B and T cell immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for the development of public health and vaccination strategies to manage the burden of COVID-19 disease. We conducted a prospective, cross-sectional analysis in COVID-19 recovered patients at various time points over a 10-month period in order to investigate how circulating antibody levels and interferon-gamma (IFN-γ) release by peripheral blood cells change over time following natural infection. From March 2020 till January 2021, we enrolled 412 adults mostly with mild or moderate disease course. At each study visit, subjects donated peripheral blood for testing of anti-SARS-CoV-2 IgG antibodies and IFN-γ release after SARS-CoV-2 S-protein stimulation. Anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies were positive in 316 of 412 (76.7%) and borderline in 31 of 412 (7.5%) patients. Our confirmation assay for the presence of neutralizing antibodies was positive in 215 of 412 (52.2%) and borderline in 88 of 412 (21.4%) patients. Likewise, in 274 of 412 (66.5%) positive IFN-γ release and IgG antibodies were detected. With respect to time after infection, both IgG antibody levels and IFN-γ concentrations decreased by about half within 300 days. Statistically, production of IgG and IFN-γ were closely associated, but on an individual basis, we observed patients with high-antibody titres but low IFN-γ levels and vice versa. Our data suggest that immunological reaction is acquired in most individuals after natural infection with SARS-CoV-2 and is sustained in the majority of patients for at least 10 months after infection after a mild or moderate disease course. Since, so far, no robust marker for protection against COVID-19 exists, we recommend utilizing both, IgG and IFN-γ release for an individual assessment of the immunity status. Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to various symptoms, including cough, fever, cold, and loss of smell and taste. The course of the disease varies in symptoms and severity, from asymptomatic infections to severe pneumonia with lung failure and death. Manifestation indices are estimated to be 55–85% . About 4SARS-CoV-2 infects human cells by using the viral spike (S) protein, which binds to the angiotensin-converting enzyme-2 (ACE-2) receptor on host cells , 4 and vIn this study, we analyzed the long-term course of the immune response with respect to serum IgG antibodies and the capacity of peripheral blood cells to produce interferon-gamma (IFN-γ) upon viral S-protein specific stimulation. On the basis of the previous experience of our group  with theThe study was performed on patients who were notified as index cases to the Health Protection Authority of the City of Luebeck/Germany [approximately 220,000 inhabitants, population density approximately 1,000/m . The stuThe data presented here were obtained from the sera of patients that were notified to the local Health Protection Authority as being SARS-CoV-2 positive by PCR irrespective of the clinical manifestation. All of them recovered from the disease without hospitalization. In total, 1,279 patients were invited by e-mail to participate. From the invited patients, 436 responded to the invitation and, finally, 412 of them were eligible for analysis after having given written informed consent and donation of blood (see flowchart). None of the study participants had received a COVID-19 vaccine.https://www.rki.de/DE/Content/Kommissionen/Stakob/Stellungnahmen/Stellungnahme-Covid-19_Therapie_Diagnose.pdf?__blob=publicationFile) and an additional category for patients who did not experience any symptoms of COVID-19 during the infection:At the study visit, a questionnaire was filled in to assess clinical comorbidities . COVID-19 disease severity was categorized based on the RKI severity definitions - moderate (signs of nonsevere pneumonia)2 < 90–94% on room air)- severe .®GENE SARS-CoV-2 RUO Test; R-Biopharm AG, Darmstadt, Germany; E-gene amplification) run on a RIDA®CYCLER according to the instruction of the manufacturer.Nasopharyngeal swabs were taken from suspected COVID-19 cases by trained personnel either in general practice or in a “drive-in” swab centre run by the Health Protection Authority between March 2020 and December 2020. Swabs were stored in stabilization media and laboratory processed within 4 h. SARS-CoV-2 RNA was detected by using an automated one-step rt-PCR (RIDAhttps://www.euroimmun.com) according to the instructions of the manufacturer. Signal-to-cut-off (SCO) ratio was calculated as the extinction value (450 nm) of the patient sample divided by the extinction level of the calibrator. A ratio between 0 and < 0.8 was considered as negative, ≥ 0.8 to < 1.1 as borderline, and a ratio ≥ 1.1 as positive. Assay specificity using pre-COVID-19 samples was calculated by the manufacturer as 100%.Serum anti-SARS-CoV-2 IgG was detected by automated enzyme-linked immunosorbent assay  was applied, through which the binding of SARS-CoV-2 S1/receptor-binding-domain RBD to ACE2 receptors of the recombinant human host cells is explored. In the first reaction step, samples and controls are incubated with soluble biotinylated ACE. If neutralizing antibodies are present in the sample, they compete with the ACE-receptor for the binding site of the SARS-CoV-2 S1/RBD proteins. Unbound ACE is removed through washing. To detect the bound ACE, a second incubation step with peroxidase-labeled streptavidin is performed, which catalyzes a color reaction. The intensity of the formed color is inversely proportional to the concentration of neutralizing antibodies in the sample. The inhibition (% IH) is calculated by the formula: % IH = 100-(extinction of patient sample × 100/extinction of blank). Values below 20 are considered negative, ≥ 20 to < 35 as borderline, and ≥ 35 as positive. According to the manufacturer, sensitivity and specificity are calculated as 95.9 and 99.7%, respectively.https://www.euroimmun.com). After 20–24 h of incubation at 37°C, the tubes were centrifuged at 12.000 rfc for 10 min. IFN-γ concentrations were measured in the supernatants by IFN-γ ELISA according to the instructions of manufacturer . When the positive control tube shows a reaction (to confirm sufficient quantity and viability of immune cells), the IFN-γ concentration from the specific stimulation tube (after subtracting the IFN-γ background) was used to quantify specific T-cell responses. Values ≥ 100 mIU/ml were interpreted as borderline, ≥ 200 mIU/ml as positive. It has to be mentioned that all IFN-γ concentrations above the measurement range were replaced by the numerical value of 2,500 mIU/ml. A further quantification was not possible.Besides B cells and antibodies, T lymphocytes and cytokines are instrumental for the shaping of the specific acute and memory immune response to SARS-CoV-2 . We sougR) and p-value were calculated to evaluate the correlation between variables, where a Person's R between 0 and 0.19 is regarded as very weak, 0.2 and 0.39 as weak, 0.40 and 0.59 as moderate, 0.6 and 0.79 as strong, and 0.8 and 1 as very strong correlation  . Of the There was a wide interindividual variation in the antibody levels, supporting the observation from our earlier study. Seropositivity was detected 16 days after the confirmed SARS-CoV-2 diagnosis by PCR . Given tn = 412). It can be clearly seen that the antibody levels decline over time. There was a moderate but significant negative linear relationship between IgG ratio and the time passed since the positive SARS-CoV-2 diagnosis . Thus, the data show that antibody expression is related to the disease severity and that antibody levels fade continuously within approximately 300 days (Table 4).To investigate factors associated with SARS-CoV-2 IgG seropositivity, two separate binary logistic regression were conducted . OverallAlthough not statistically significant, a tendency for a greater OR of being seropositive was seen among participants having at least one comorbidity  or were of advanced age . For female participants, the OR for being seropositive was almost equal to males  suggesting that being seropositive is equally likely to occur in both female and male participants.We also investigated factors associated with SARS-CoV-2 neutralizing seropositivity conducting two separate logistic regression analyses . We founr = 0.6, p < 0.001).Since T cells are instrumental for the development of the S-protein reactive B-cell activity, we explored T-lymphocyte activity. We chose to investigate the induction and release of IFN-γ upon S-protein specific stimulation. This assay reflects an easy-to-perform summarized image of T-lymphocytes activity without considering all subgroups of T-lymphocytes or other IFN-γ-producing cells. As can be seen in Looking at the correlation between antibody levels and IFN-γ concentrations, a heterogeneous picture emerged . While ivia PCR from a nasopharyngeal swab. The antibody levels continuously decreased over time.In summary, in our SARS-CoV-2 PCR-positive cohort, the antibody profile was heterogeneous. In most cases, antibodies could be detected between 16 and 73 days after the determination of a SARS-CoV-2 infection In 65 of 412 patients (15.8%), no significant antibody levels could be detected in two up to four consecutive analyses between days 16 and 73. As far as the IFN-γ release is concerned, a similar picture emerged. The levels were heterogeneous and decreased over time. Noteworthy, a significant part of the patients produced very high levels of IFN-γ irrespective of the concentration of antibodies measured .In this study, we evaluated the development of specific humoral and cellular immune responses in outpatients recovered from SARS-CoV-2 infection, mostly with mild-to-moderate disease  in a croIn this study, about 16% of the patients had neither positive nor borderline detectable IgG antibodies at the last study visit. These data substantiate our previous findings from the first wave of the pandemic in early 2020 . On a poAt the time the study was done, no variants were detected in the area of Luebeck. We are therefore sure that our neutralization assays did not fail due to undetected virus variants.Very few published data sets compare antigen-specific B-cell and T-cell immunity. We, therefore, examined interrelationships between IgG antibody levels and IFN-γ release. Like with the antibody kinetics, the IFN-γ values decayed over time after infection with similar kinetics  which isSince our data and those from others show that determination of antibodies alone is not predictive for protection against SARS-CoV-2 disease, simultaneous determination of IFN-γ may be a valuable adjunct and may also predict the time-point for possible necessary booster vaccinations on an individual basis. The antigens used are based on the S1 of the wild type—this is the antigenic origin currently used by all well-known manufacturers. We mainly conclude from our data that using antibody, neutralizing and IFN-γ assays are useful to monitor individual immune responses. However, we know that in infections with the beta or delta variant, for example, there are mutations that lead to a kind of immune escape. Theoretically, it cannot be ruled out that in infections with highly divergent variants, differences in immune responses against the S1 used would be observed.This study has few limitations that must be acknowledged. Longitudinal data for each subject, with at least three time-points per subject, would be required to better understand the kinetics of durability of SARS-CoV-2-specific antibodies. Furthermore, since patients were sampled only once at various time-points after the confirmed COVID-19 diagnosis, the results are vulnerable to bias caused by individual variations and inter-individual variation cannot be excluded. Nevertheless, the current cross-sectional data describe well the dynamics of spike-specific antibodies over 10 months and IFN-γ release by blood T lymphocytes at one study point. This study was not sufficiently powered to control for many variables simultaneously.This study shows that on average 9.8 months after detecting SARS-CoV-2 in nasopharyngeal specimen, we found a significant weaning of IgG antibodies levels against the viral S-protein in comparison to the initial values. Most of the patients showed robust IFN-γ production after S-protein stimulation of peripheral blood cells, indicating the importance of T lymphocytes for shaping the protective immune reaction. However, in a substantial proportion of our samples, we found low-antibody levels accompanied with high-IFN-γ levels and vice versa. For future assessment of protection and possible vaccination strategies, both, determination of antibodies and IFN-γ is recommended.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The ethical committee of the University of Luebeck approved the study (ref. 20-339). The patients/participants provided their written informed consent to participate in this study.JS, IB, JR, AM, and WS wrote the article. JS, IB, DZ, JK, and WS were responsible for data analysis. SS, TM, and JR were responsible for the recruitment of patients, data collection, and contributed to data analysis. AM and WS designed the study. All authors approved the final version of the manuscript.This study was funded by the City of Luebeck and EUROIMMUN AG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.